Johne's disease (paratuberculosis) is a chronic wasting disease of ruminant animals caused by the bacterium Mycobacterium avium subspecies paratuberculosis (M. ptb).
The disease spreads insidiously, with animals becoming infected early in life following ingestion of contaminated milk, collostrum, or pasture. In many countries, including New Zealand and the USA, herds are not routinely tested for M. ptb infection. The number of herds officially known to have infection is therefore thought to be a gross underestimate of the actual level of infection. Estimates suggest that approximately 60% of dairy herds in New Zealand are infected with M. ptb. In sheep, prevalence of M. ptb infection may be as high as 70%.
Infected animals are less productive, which results in significant economic losses for farmers. The cost to New Zealand farmers is estimated to be in excess of $30 million per annum. The economic impact in the USA was estimated to be as much as US$1.7 billion per annum (Chiodini et al., 1984a).
Good management practices, herd testing, and culling of infected animals are important tools for controlling paratuberculosis. However, this strategy alone is unlikely to completely control the problem because current diagnostic tests frequently fail to identify infected animals in the early, preclinical stages of disease. The early stages of disease are asymptomatic and shedding of the organism in the faeces is undetectable or intermittent. Only animals which progress to the terminal stage generally show clinical disease, and that only after two to five years (Stuart, 1965; Lepper et al., 1989). For this reason, it is suggested that for every clinical case on a farm, there are approximately 20 additional infected animals.
Detection of infection in the host is influenced by the stage of disease (Ridge et al., 1991; Sweeney et al., 1995). In the lengthy preclinical stage, the bacterium produces little or no detectable immune response and the number of organisms is typically low, making direct detection of the bacterium difficult. In cattle, culture of M. ptb from faeces or tissue is currently the most accurate means of detecting infection. The success of culture is linked to the presence of sufficient numbers of M. ptb being shed from the intestine. Hence, animals in the early stages of disease, which do not shed the bacterium, or shed intermittently, are difficult to detect by this method. A further disadvantage of culture is the long incubation time required for the appearance of colonies.
Serological testing is also available, however, this performs best in animals with clinical disease (80% detection) and poorly with preclinically infected animals (as low as 15% detection), (Billman-Jacobe et al., 1992; Sweeney et al., 1995).
Alternatively, M. ptb can be confirmed through PCR to identify the presence of the species-specific DNA fragments. To date, only three subspecies-specific DNA fragments have been identified in the M. ptb genome.
Of these, the most widely used is the IS900 element. IS900 detection by PCR is available as a commercial kit (Idexx, USA). This test is reported to have a sensitivity of approximately 60% in infected cattle, based on cattle diagnosed by faecal culture (Whipple et al., 1992). A report of finding organisms that give positive results with IS900 PCR but are not M. ptb has placed some doubt on the routine use of this test as the ultimate confirmation of M. ptb (Cousins et al., 1999).
Eradication of Johne's disease is difficult due to the inability of current diagnostic tests to detect all infected animals. As a consequence, more animals are put at risk of infection because preclinically infected animals intermittently shed the bacterium, thereby spreading the organism. Therefore, improvements in the sensitivity of diagnostic testing and/or increased immunity of uninfected animals would be beneficial.
Commercially available vaccines for the control of Johne's disease contain whole organisms, either attenuated strains of M. ptb (eg. Neoparasec—Medal, France), or heat-killed preparations (eg. Gudair—CZ Veterinaria, Porrino, Spain), which are mixed with an oily adjuvant and injected subcutaneously. Vaccination does not prevent or eliminate infection, but reduces the number of animals that progress to clinical disease and the excretion of organisms in the faeces.
Unfortunately, these whole cell vaccines come with a number of drawbacks. Firstly, the immunogenic load presented by the whole cell in conjunction with the adjuvant induces a severe hypersensitivity reaction at the injection site, which can cause the formation of a persistent nodule (granuloma). Occasional rupture of the nodule causes suffering to the animal and potential downgrading of the carcass at slaughter, with concomitant diminished returns for the farmer. Histological examination of nodules or regional lymph nodes can reveal the presence of acid-fast organisms that can be confused with the tuberculosis organism. Additionally, while Johne's disease is distinct from tuberculosis, and caused by a distinct organism, the current Johne's vaccines can generate cross-reactive responses to M. bovis skin test antigens which can interfere with tuberculosis control programmes.
Accordingly, there is a need for alternative methods for detecting Johne's disease in animals, and proteins and/or markers and/or vaccines useful in such methods.
The applicants have identified and characterized a novel protein, from M. ptb, which exhibits strong potential as a diagnostic marker and a subunit vaccine. A raw nucleotide sequence similar to that encoding the protein of the invention was located in the TIGR Mycobacterium avium subspecies avium database. This nucleotide database was unannotated, the sequence had not been identified as an open reading frame, nor as a gene, and no protein-encoding utility had been ascribed to the raw nucleotide sequence. Accordingly, the applicants are the first to teach the identification of this nucleotide sequence as a protein-encoding gene. The protein can be readily obtained from a culture filtrate of M. ptb, or expressed in a heterologous host, as the gene encoding this protein has also been identified and characterized by the applicants. It is towards this protein that the present invention is broadly directed.